QUANTITATION OF INTERMEDIARY METABOLISM IN HEPATOCYTES

与基质孵育的肝细胞中碳流的现实模型 含有葡萄糖、核糖、果糖、丙氨酸和乙酸盐的混合物已经被 开发并用于获得代谢物流的定量描述 在胰高血糖素存在和不存在下。 为了增进我们对 肝细胞中间代谢结构组织，我们 将进行配对实验（在存在和不存在 加压素），其中天冬氨酸和谷氨酸将被添加到上述 底物混合物。 这些实验不仅将提供进一步的 关于处理致炎性和致脲性氨基酸的信息，但 将允许加压素的作用与那些 胰高血糖素对代谢物沿主要血管沿着流动的整个模式的影响 中间代谢途径。 我们还打算孵化 从禁食大鼠中分离的肝细胞与含有以下物质的底物混合物 芥酸、油酸、十二烷酸、己酸、丙酸、乙酸、乳酸和 丙酮酸，其中一种底物标记有14 C，在任何给定的 烧瓶 约80个独立测量的标签流入CO2， 葡萄糖，和乙酰乙酸的1和2，3，4位将被制备， 数量足够超过独立通量参数的数量 以产生对所提出的脂肪酸模型的严格检验 代谢，从而定量估计β-氧化在 线粒体和过氧化物酶体途径和欧米茄氧化的每一个 混合物中的脂肪酸。 这些研究将扩展：1） 使用从用苯扎贝特治疗的大鼠中分离的肝细胞， 已知可增加过氧化物酶体β-氧化酶降血脂药; 和2）使用含有葡萄糖、核糖、果糖 丙氨酸、乙酸、谷氨酸、油酸和芥酸，以获得 代谢物流的定量描述沿着的途径， 上面开发的模型。

A realistic model of carbon flow in hepatocytes incubated with a substrate mixture containing glucose, ribose, fructose, alanine, and acetate has been developed and used to obtain a quantitative description of metabolite flows in the presence and absence of glucagon. To increase our understanding of the structural organization of intermediary metabolism in hepatocytes, we will carry out paired experiments (in the presence and absence of vasopressin) in which aspartate and glutamate will be added to the above substrate mixture. These experiments will not only provide further information on the handling of gluconeogenic and ureagenic amino acids, but will allow quantitative comparison of the effects of vasopressin with those of glucagon on the entire pattern of metabolite flow along the major pathways of intermediary metabolism. We also intend to incubate hepatocytes isolated from fasted rats with a substrate mixture containing erucoate, oleate, dodecanoate, hexanoate, propioante, acetate, lactate, and pyruvate, with one of these substrates labeled with 14C in any given flask. Approximately 80 independent measurements of label flow into CO2, glucose, and the 1 and 2, 3, 4 positions of acetoacetate will be made, a number sufficiently in excess of the number of independent flux parameters to be computed to yield a stringent test of a proposed model of fatty acid metabolism and thus a quantitative estimate of Beta-oxidation in the mitochondrial and peroxisomal pathways and of Omega-oxidation for each of the fatty acids in the mixture. These studies will be extended by: 1) using hepatocytes isolated from rats treated with bezafibrate, a hypolipidemic drug known to increase peroxisomal Beta-oxidation enzymes; and 2) using a substrate mixture containing glucose, ribose, fructose, alanine, acetate, glutamate, oleic, and erucic acids to obtain a quantitative description of metabolite flow along the pathways of the combined models developed above.